In human and other mammalian cells, pre-mRNA is packaged by six abundant nuclear proteins to form a repeating array of regular ribonucleoprotein particles (40S hnRNP particles). RNA packaging is the first of several physical-chemical events involved in RNA maturation. It preexists and accommodates the biochemical events of intron recognition and excision, exon ligation, base modification, and transport to the cytoplasm. Few of these events are likely to be understood in the absence of a detailed knowledge of the RNA packaging mechanism. Several recent breakthroughs have revealed that the 40S core particle is composed of 3 copies of three different protein tetramers and a 700 +/- 20nt fragment of pre-mRNA. In addition, it is now possible to purify the six core particle proteins under non-denaturing conditions and study their structures and their in vitro and in vivo interactions. The long term objective of the studies described here is to elucidate the fundamental interactions of the core particle proteins which lead to particle assembly and to particle function in RNA metabolism. To generate definitive new information on the topology of protein and RNA in the 40S hnRNP core particle, emphasis will focus on a detailed molecular characterization of several intermediate structures formed during the controlled in vitro assembly of 40S monoparticles from purified components. Additional structural information will come from an ultrastructural characterization of the assembly intermediates and from electron and X-ray diffraction studies on crystals and fibers formed by two of the purified tetramers. Specific Aims 1-4 will provide structure-relevant biochemical and physical chemical information while Specific Aims 5 and 6 will provide direct structural information (EM and crystallographic) required to confirm or exclude deductions from the biochemical studies. The basic knowledge derived from these studies exists as a prerequisite to a complete understanding of the mechanisms of several developmental disorders and genetic-based diseases which result form abnormal RNA maturation. In addition, this knowledge will be important in understanding the mechanisms which drive alternative RNA splicing and its role in the generation of antibody diversity, the IgM/IgD switch, and other immune system functions.